Anti-cancer and Neuroprotective Effects of Conjugated Graphene Quantum Dots (GQDs) in Brain Tumor Bearing Animal Model

Abstract

Glioblastoma (GBM), has been recognized as a most perilous and highly malignant type of primary brain tumor. It has a dismal prognosis and only 21 months of average survival rate after combinatory multimodal therapies included chemotherapy, surgery, and radiotherapy. Due to the infiltrative and intrusive nature of GBM tumor-microenvironment (TME), it is essential to design elective therapy for specific targeting of tumor cells. A various approach has been made to upgrade in-vitro and in-vivo models with a prime goal to assessing molecular mechanisms which would be suitable targets to design novel nano-onco-therapeutics. With this prime inspiration, we have developed a Graphene-based fluorescence 2-D quasi-spherical tiny quantum dot which has a particle size less than 10 nm, through bottom-up techniques which has the simplest, efficient, high yield, low-cost and less time-consuming processes. Synthesis of Graphene quantum dots (GQDs) was done by hydrothermal and direct pyrolysis of carbohydrates precursors at relatively high temperatures. The narrow-size distribution of less than 20 nm of prepared GQD was characterized by DLS, TEM, AFM. The larger diameter of GQD was obtained in DLS due to the hydrodynamic effect of an aqueous solution. Spectro-fluorescence analysis was shown that GQD has size-dependent fluorescence emission. 13C-NMR spectroscopy studies suggested that carbonyl ester and carboxylic functional groups were present on the surface edge of the GQD. For surface activation and functionalization of GQDs with three bio-recognized protein/antibodies such as BSA, Caspase 8 and Trastuzumab were being developed by two different approaches: those were 1) PEGylation and 2) Carbodiimide activated amidation. Gel electrophoresis, FTIR, Spectro-fluorescence, and DLS were performed to characterize the developed conjugations. From the results, we concluded that carbodiimide activated amidation reaction was successful and made stable conjugation of GQD-protein/antibodies as compared PEGylated process. Work was also carried out in the structural-based in-silico molecular dynamic simulation and virtual screening analysis by AutoDock, PyRx, Discovery studio visualizer. Based on the post-screening analysis and higher negative energy integration indicated that conjugation was stable and formed rapidly. In-vitro % cell viability assay was performed in two different brain cancer cell lines: 1) SK-N-SH and 2) N2a. GQDs was shown dose-dependent cytotoxicity on both cell lines. The pure GQDs was exhibited lethal toxicity around the concentration of 50 μg/ml, but the same concentration showed decreasing in toxicity upon conjugations. In-vitro scratch assay in SK-N-SH cell line indicated that the same concentration showed an increase in wound closure activity as compared to lower concentration in a similar time of exposure.% Hemolysis of RBC and cell ruptured was observed at GQDs concentration 100 μg/cubic cell/ml. The rat serum and plasma CRP levels were increased at a dose concentration above 1000 mg/kg body weight. A newer animal brain tumor model was developed by chemical induction of Ethyl-nitroso urea (ENU) through different routes: 1) IP, 2) Indirect bregma and 3) Indirect Lambda focal administrations. In which, lambda focal point administration animal group was showed rapid tumor progression. GQD-Caspase 8 conjugation from the all-over conjugations was shown significant anti-glioma and neuroprotective activity in developed brain tumor-bearing animal models.